Ex vivo method for determining potential glp-2 receptor modulations

ABSTRACT

Disclosed herein is a method for measuring the contractility of intestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Also disclosed is an assay which directly measures the activity of GLP-2 or GLP-2 ligands ex vivo and permits the screening of putative GLP-2 ligands in native tissue.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/741,075 filed on Dec. 1, 2005, the entirecontents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Glucagon-like peptide-2 (GLP-2) is a 33-amino acid proglucagon-derivedpeptide secreted by the endocrine L-cell primarily in the lowergastrointestinal tract in response to luminal nutrients. Plasma levelshave been shown to significantly increase within an hour of ingesting ameal, in particular following the ingestion of carbohydrates or fat (1).GLP-2 has been shown to be responsible for the regulation ofproliferation and apoptosis of the intestinal epithelium (2,3). Thesechanges in part result in an increase in mucosal surface area, enhancedabsorptive efficiency and barrier function in the small intestine (4-6).GLP-2 also decreases gastric motility, inhibits gastric acid secretion,increases nutrient transport activity and acutely increases intestinaland portal blood flow (7-11).

GLP-2 and GLP-2 analogues promote the growth and repair of theintestinal epithelium in models of disease, including enhancedadaptation and nutrient absorption following small bowel resection andalleviation of TPN-induced hypoplasia in rodents (12-15). GLP-2analogues have demonstrated decreased mortality and improvement ofdisease-related histopathology in animal models of intestinal damagesuch as indomethacin-induced enteritis, dextran sulfate-induced colitisand chemotherapy-induced mucositis (16-19).

The intestinotrophic effects of GLP-2 are mediated by the GLP-2 receptor(GLP-2R), a member of the superfamily of G-protein coupled receptors andmost closely related to the GLP-1 and glucagon receptor gene subfamily(20). The GLP-2R is a high-affinity, ligand- specific functionalreceptor coupled to the G-protein Gs. Studies of the activation of thecloned GLP-2R by GLP-2 analogues show a correlation of in vitro activitywith in vivo intestinotrophic efficacy (20).

To date, characterization of GLP-2R function has been limited toheterologous cell line expression, mucosal fractions and primary cellcultures. GLP-2 selectively stimulates cAMP production in recombinantlyexpressing GLP-2R cell lines, isolated intestinal mucosal fractionscontaining enteroendocrine and neural cells and primary hippocampalcultures (21-24). The study of native GLP-2R biological activity hasfocused on in vivo animal models and ex vivo model systems that requireexogenous application of GLP-2 to the whole animal prior to tissueisolation (2-19, 25-31). There is no ex vivo method for characterizingputative GLP-2 ligands directly; such a method would have obviousutility, for example in the screening and characterization of GLP-2ligands of pharmaceutical interest.

SUMMARY OF THE INVENTION

Disclosed herein is a method for measuring the contractility ofintestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Thus, oneaspect of the invention is an assay which directly measures the activityof GLP-2 or GLP-2 ligands. Another aspect of the invention is the directcharacterization of the GLP-2 receptor. A further aspect of theinvention is a method for screening compounds for activity at the GLP-2receptor, such as putative GLP-2 agonists, antagonists, modulators orthe like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the organ bath trace of the effect of 1 uM GLP-2(1-33) onrat contractility in duodenum, jejunum, ileum and colon.

FIG. 2 shows the organ bath trace of the effect of GLP-2(1-33) on ratcolon contractility.

FIG. 3 Shows the organ bath trace of the effect of [gly2]GLP-2 on ratcolon contractility.

FIG. 4 shows the inhibitory effects of GLP-2 analogues on spontaneouscontractility in rat colon.

FIG. 5 shows the effect of GLP-2 analogues on cAMP accumulation inEBNA293 cells stably expressing rat GLP-2R.

FIG. 6 shows the effect of GLP-2(3-33) on GLP-2(1-33)-induced inhibitionof colon contractility.

FIG. 7 shows the effect of GLP-2(3-33) on teduglutide-induced inhibitionof rat colon contractility.

FIG. 8 shows the effect of a small molecule antagonist onGLP-2(1-33)-induced inhibition of colon contractility.

FIG. 9 shows the effect of a small molecule positive modulator onGLP-2(1-33)-induced cAMP accumulation.

FIG. 10 shows the effect of a small molecule positive modulator onGLP-2(1-33)-induced inhibition of colon contractility.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

hGLP-2R is the human GLP-2 receptor; rGLP-2R is the rat GLP-2 receptor.hGLP-2 is human GLP-2 (also known as Teduglutide); rGLP-2 is rat GLP-2.

A “GLP-2 ligand” is defined as any molecule which interacts with theGLP-2 receptor; such molecules may be peptides (such as analogues ofnaturally occurring GLP-2) or small molecules.

A “modulator” is defined as a molecule which modulates the activity of aGLP-2 agonist.

“Intestinal tissue” is broadly defined as intestine from the pylorus tothe rectum. “Small intestine” is defined as intestine from the pylorusto the ileo-cecal valve (it may also be defined as duodenum, jejunum andileum). “Large intestine” is defined as intestine distal from the cecum(it may also be defined as colon tissue).

Disclosed herein is an assay which, for the first time, demonstrates theability of GLP-2 receptor ligands to directly affect nativeGLP-2R-mediated effects in colon tissue ex vivo. As a result, it ispossible to directly screen and pharmacologically characterize GLP-2ligands, such as peptidic analogies of GLP-2, as well as small moleculeagonists, antagonists and modulators, against whole tissue.

Such tissue is of mammalian origin; in one aspect it is of rat origin,in others it is of mouse or guinea-pig origin.

The assay method comprises measuring the contractility of segments ofintestinal tissue ex vivo. The fact that this assay is useful for thestated purpose is not obvious, based upon the known expression levels ofthe GLP-2 receptor in this tissue. Further, previous studies have shownthat direct application of GLP-2 ex vivo to this tissue has no effect.

One aspect of the invention is a method for characterizing the GLP-2receptor; for example in an endeavour to better understand thephysiological role of GLP-2 and the GLP-2 receptor in intestinal tissueor to elucidate the role of putative mediators related to GLP-2activation and physiology.

Another aspect of the invention is a method for determining whether acompound is active on the GLP-2 receptor comprising the step ofmeasuring contractility of intestinal tissue segments ex vivo in thepresence and absence of compound, wherein a difference in contractilityin the presence of the compound is indicative of activity on the GLP-2receptor.

Yet another aspect of the invention is a method for determining whethera compound acts as a GLP-2 receptor agonist comprising the step ofmeasuring contractility of intestinal tissue segments ex vivo in thepresence and absence of compound, wherein inhibition of contractility inthe presence of the compound is indicative of GLP-2 receptor agonistactivity.

Yet another aspect of the invention is a method for determining whethera compound acts as a GLP-2 receptor antagonist comprising the step ofmeasuring the effect of a GLP-2 receptor agonist on contractility ofintestinal tissue segments ex vivo in the presence and absence of thecompound, wherein a decrease of agonist inhibition of contractility inthe presence of the compound is indicative of GLP-2 receptor antagonistactivity.

Another aspect of the invention is a method of assaying compounds foractivity at the GLP-2 receptor comprising the steps of:

i. Obtaining a segment of intestinal tissue,

ii. Suspending the segment in an organ bath,

iii. Placing the segment under tension,

iv. Incubating the segment with a compound of interest and

v. Measuring the contractility of the tissue; wherein an effect oncontractility of the segment is indicative of activity on the GLP-2receptor.

In one aspect the tissue is intestinal tissue. In another it is smallintestine tissue. In yet another it is large intestine tissue or colontissue. FIG. 1 shows the Organ Bath Trace of the effect of 1 uMGLP-2(1-33) on rat contractility in duodenum, jejunum, ileum and colon.FIG. 2 shows that GLP-2(1-33) inhibits spontaneous colon contractilityin a concentration-dependent manner in rat colon tissue; FIG. 3 showsthe analogous effect of Teduglutide.

The assay of putative agonists may be conducted by incubation of thecolon segments with a predetermined concentration of the ligand to betested for 5 to 10 minutes. FIG. 4 shows typical results for a number ofGLP-2 analogues. FIG. 5 shows the effect of GLP-2 analogues on cAMPaccumulation in EBNA293 cells stably expressing rGLP-2R.

The assay of putative antagonists may be conducted by pre-incubation ofthe colon segments with a predetermined concentration for, for example,5 to 10 minutes of the ligand to be tested followed by addition andincubation of a predetermined concentration of a GLP-2 agonist. FIGS. 6,7 and 8 show typical results for a number of GLP-2 antagonists;concentration-response curves are clearly right-shifted, as expected.

The assay of putative modulators, may be conducted by pre-incubation ofthe colon segments with a predetermined concentration of the ligand tobe tested for, for example, 5 to 10 minutes followed by the addition andincubation of a pre-determined concentration of a GLP-2 agonist. FIG. 10show typical results for a GLP-2 positive modulator; the effect of themodulation is clearly demonstrated. FIG. 9 shows the correspondingeffect on GLP-2(1-33)-induced cAMP accumulation.

METHODS & RESULTS

It should be noted that the methods described below are illustrative innature, and that the invention is not limited to the particularembodiments described.

Segments of colon from male Sprague Dawley rats were suspended in organbath chambers containing Krebs' solution and maintained at 37° C. with95% O₂ and 5% CO₂. Basal tone, contractility rate and peak height wereused to measure the properties of spontaneous and ligand-mediatedcolonic contractions. Segments were initially loaded to a tension of 2 gand allowed to equilibrate for 60 minutes. During this period, segmentswere repeatedly washed every 10 minutes. [gly2]GLP-2 (“Teduglutide”, aGLP-2 agonist) and GLP-2 analogues were subsequently incubated with eachtissue segment for 5 to 10 minutes. The tissues were allowed tore-equilibrate with repeated washes following each experiment.

Teduglutide and GLP-2(1-33) inhibited muscle contractility in aconcentration-dependent manner with an estimated IC₅₀ of 4.9±1.9 (SE) nM(n=6) and 13.5±5.4 nM (n=9), respectively. While having no effect alone,GLP-2(3-33) (a GLP-2 antagonist/partial agonist) blocked the inhibitoryeffects of Teduglutide and GLP-2. In the presence of 10 μM GLP-2(3-33),Teduglutide and GLP-2(1-33) inhibited colon contractility with an IC₅₀of 59.9±11.9 nM (n=6) and 142.7±55.9 nM (n=5) respectively. The rankorders of potency of GLP-2 peptide analogues were similar to thatpreviously reported for their cAMP activation in cell linesrecombinantly expressing the rGLP-2R.

These studies demonstrate the ability of GLP-2 to reproducibly andsignificantly reduce spontaneous contractility of rat colon segmentssuspended in organ bath chambers. GLP-2-induced inhibition of coloncontractility is concentration-dependent and reversible following awash-out period. The GLP-2R antagonist/partial agonist GLP-2(3-33)blocks the effect of GLP-2(1-33) in a concentration-dependent manner.The rank order of potency of GLP-2 analogues on the inhibition of coloncontractility was comparable to the rank order of increased cAMPaccumulation in heterologous cells expressing GLP-2R.

Therefore the described assay enables native GLP-2R characterization bythe direct application of GLP-2 to intestinal tissue segments ex vivo.As a result, it is possible to screen and pharmacologically characterizeGLP-2 peptide analogues, GLP-2R small molecule agonist and antagonistsagainst whole tissue.

Colon Contractility—Methods

Animals

Male Sprague-Dawley (SD) rats (Charles River Canada, Quebec), weighing200-250 g, were used for these studies. The animals were housed oncontact bedding and kept with free access to food (Purina Rodent Diet)and water in a facility with regulated temperature and humidity, underan alternate 12-hour light/dark cycle. Animals were acclimatized for 3-5days prior to harvesting of tissues.

Tissue Preparation

Rats were euthanized by decapitation. The entire intestine was quicklyremoved and placed in a Petri dish containing a Sylgard layer and 37° C.Krebs' solution bubbled with 95% O₂ and 5% CO₂. The Krebs' solution (pH7.4) contained 118.1 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄,1.2 mM MgSO₄.7H₂O, 2.5 mM CaCl₂.2H₂O and 11 mM glucose. The colon wasisolated, cleaned of superficial fat and mesentery and sectioned intoeight 1-cm long segments. The lumen was cleaned by carefully flushingeach segment with Krebs' solution using plastic transfer pipets.Segments were transferred to a separate Petri dish containing freshKrebs' solution and suture threads were tied to both ends of eachsegment. Tissues were suspended longitudinally using surgical suture,one end attached to the transducer on the top and the other to thetissue holder at the bottom of 20 ml organ bath chambers containingKrebs' solution, maintained at 37° C. with 95% O₂ and 5% CO₂. Isometrictensions were continuously measured using calibrated force displacementtransducers and data was collected and processed by the MP100WSW systemwith AcqKnowledge software

Experiment Protocol

Segments of colon were initially loaded to a tension of 2 g and allowedto equilibrate for a 60 min period. During this period, segments werewashed with Krebs' solution at 10 minutes intervals. When the tissuesobtained regular and stable contraction patterns, GLP-2 agonist responseexperiments were conducted by the addition of eight concentrations ofcompound into eight separate organ baths for 5-10 minutes. Routinely,one-hour washes were interspersed between repeat applications ofcompounds to allow for recovery. The effect of GLP-2(3-33) on GLP-2agonist analogues were conducted by pre-incubating colon segments with 1or 10 μM GLP-2(3-33) for 5-10 minutes. Then agonist responses weremeasured at various concentrations as described above.

Data Analysis

Tension was measured by changes in peak height. Data were calculated andexpressed as percentage response relative to the basal level (prior tothe experiment). Graphs and IC₅₀ determinations were plotted by thepercentage response in GraphPad Prism (v 3.02). In order to assess thesignificance of the effect of GLP-2 and Teduglutide paired t-tests wereconducted on the Log [IC₅₀] values.

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1. A method for determining whether a compound is active on the GLP-2receptor comprising the step of measuring contractility of intestinaltissue segments ex vivo in the presence and absence of compound, whereina difference in contractility in the presence of the compound isindicative of activity on the GLP-2 receptor.
 2. A method according toclaim 1 wherein the intestinal segments are of mammalian origin.
 3. Amethod according to claim 2 wherein the intestinal segments are of humanorigin.
 4. A method according to claim 2 wherein the segments are of ratorigin.
 5. A method according to claim 1 wherein the tissue is smallintestine tissue.
 6. A method according to claim 1 wherein the tissue iscolon tissue.
 7. A method for determining whether a compound acts as aGLP-2 receptor agonist comprising the step of measuring contractility ofintestinal tissue segments ex vivo in the presence and absence ofcompound, wherein inhibition of contractility in the presence of thecompound is indicative of GLP-2 receptor agonist activity.
 8. A methodaccording to claim 7 wherein the tissue is rat colon tissue.
 9. A methodaccording to claim 7 wherein the tissue is human colon tissue.
 10. Amethod for determining whether a compound acts as a GLP-2 receptorantagonist comprising the step of measuring the effect of a GLP-2receptor agonist on contractility of intestinal tissue segments ex vivoin the presence and absence of the compound, wherein a decrease ofagonist inhibition of contractility in the presence of the compound isindicative of GLP-2 receptor antagonist activity.
 11. A method accordingto claim 10 wherein the GLP-2 receptor agonist is GLP-2.
 12. A methodaccording to claim 10 wherein the GLP-2 receptor agonist is a GLP-2analogue.
 13. A method according to claim 12 wherein the GLP-2 analogueis teduglutide.
 14. A method according to claim 10 wherein the tissue iscolon tissue.
 15. A method according to claim 14 wherein the tissue israt colon tissue.
 16. A method for determining whether a compound isactive on the GLP-2 receptor comprising the steps of i. Obtaining asegment of intestinal tissue, ii. Suspending the segment in an organbath, iii. Placing the segment under tension, iv. Incubating the segmentwith the compound and v. Measuring the contractility of the tissue;wherein an effect on contractility of the segment is indicative ofactivity on the GLP-2 receptor.
 17. A method according to claim 16wherein the tissue is human colon tissue.
 18. A method according toclaim 16 wherein the tissue is rat colon tissue.